The transforming growth factor beta (TGFβ) signaling pathway is involved in many cellular processes in both the adult organism and the developing

embryo An embryo ( ) is the initial stage of development for a multicellular organism. In organisms that reproduce sexually, embryonic development is the part of the life cycle that begins just after fertilization of the female egg cell by the male sp ...

including

cell growth Cell most often refers to: * Cell (biology), the functional basic unit of life * Cellphone, a phone connected to a cellular network * Clandestine cell, a penetration-resistant form of a secret or outlawed organization * Electrochemical cell, a de ...

cell differentiation Cellular differentiation is the process in which a stem cell changes from one type to a differentiated one. Usually, the cell changes to a more specialized type. Differentiation happens multiple times during the development of a multicellular ...

, cell migration,

apoptosis Apoptosis (from ) is a form of programmed cell death that occurs in multicellular organisms and in some eukaryotic, single-celled microorganisms such as yeast. Biochemistry, Biochemical events lead to characteristic cell changes (Morphology (biol ...

, cellular homeostasis and other cellular functions. The pathway is also involved in multiple physiological processes such as regulation of the immune system, the vascular system and embryonic development. The TGFβ signaling pathways are conserved. In spite of the wide range of cellular processes that the TGFβ signaling pathway regulates, the process is relatively simple.

TGFβ superfamily The transforming growth factor beta (TGF-β) superfamily is a Protein superfamily, large group of structurally related cell regulatory proteins that was named after its first member, TGF beta 1, TGF-β1, originally described in 1983. They interac ...

ligands bind to a type II receptor, which recruits and

phosphorylates In biochemistry, phosphorylation is described as the "transfer of a phosphate group" from a donor to an acceptor. A common phosphorylating agent (phosphate donor) is ATP and a common family of acceptor are alcohols: : This equation can be writt ...

a type I receptor. The type I receptor then phosphorylates receptor-regulated SMADs (

R-SMAD R-SMADs are receptor-regulated SMADs. SMADs are transcription factors that transduce extracellular TGF-β superfamily ligand signaling from cell membrane bound TGF-β receptors into the nucleus where they activate transcription TGF-β target ge ...

s) which can now bind the coSMAD

SMAD4 SMAD4, also called SMAD family member 4, Mothers against decapentaplegic homolog 4, or DPC4 (Deleted in Pancreatic Cancer-4) is a highly conserved protein present in all metazoans. It belongs to the SMAD family of transcription factor proteins ...

. R-SMAD/coSMAD complexes accumulate in the nucleus where they act as

transcription factor In molecular biology, a transcription factor (TF) (or sequence-specific DNA-binding factor) is a protein that controls the rate of transcription (genetics), transcription of genetics, genetic information from DNA to messenger RNA, by binding t ...

s and participate in the regulation of target gene expression.

Mechanism

Ligand binding

The TGF beta superfamily of ligands includes: bone morphogenetic proteins (BMPs), growth and differentiation factors (GDFs), anti-Müllerian hormone (AMH),

Activin Activin and inhibin are two closely related protein complexes that have almost directly opposite biological effects. Identified in 1986, activin enhances FSH biosynthesis and secretion, and participates in the regulation of the menstrual c ...

Nodal Nodal may refer to: * Nodal, the adjectival form of the noun :wikt:node * Nodal homolog, a protein encoded by the gene NODAL and responsible for left-right asymmetry * Nodal (software), a novel music composition program * Christian Nodal ( ...

and TGFβs. Signaling begins with the binding of a TGF beta superfamily ligand to a TGF beta type II receptor. The type II receptor is a serine/threonine receptor kinase, which

catalyzes Catalysis () is the increase in rate of a chemical reaction due to an added substance known as a catalyst (). Catalysts are not consumed by the reaction and remain unchanged after it. If the reaction is rapid and the catalyst recycles quick ...

the

phosphorylation In biochemistry, phosphorylation is described as the "transfer of a phosphate group" from a donor to an acceptor. A common phosphorylating agent (phosphate donor) is ATP and a common family of acceptor are alcohols: : This equation can be writ ...

of the Type I receptor. Each class of ligand binds to a specific type II receptor. In mammals there are seven known type I receptors and five type II receptors. There are three activins:

Activin A Activin and inhibin are two closely related protein complexes that have almost directly opposite biological effects. Identified in 1986, activin enhances FSH biosynthesis and secretion, and participates in the regulation of the menstrua ...

, Activin B and Activin AB. Activins are involved in embryogenesis and osteogenesis. They also regulate many

hormones A hormone (from the Greek participle , "setting in motion") is a class of signaling molecules in multicellular organisms that are sent to distant organs or tissues by complex biological processes to regulate physiology and behavior. Hormones a ...

including

pituitary The pituitary gland or hypophysis is an endocrine gland in vertebrates. In humans, the pituitary gland is located at the base of the brain, protruding off the bottom of the hypothalamus. The pituitary gland and the hypothalamus control much of th ...

, gonadal and

hypothalamic The hypothalamus (: hypothalami; ) is a small part of the vertebrate brain that contains a number of nuclei with a variety of functions. One of the most important functions is to link the nervous system to the endocrine system via the pituit ...

hormones as well as

insulin Insulin (, from Latin ''insula'', 'island') is a peptide hormone produced by beta cells of the pancreatic islets encoded in humans by the insulin (''INS)'' gene. It is the main Anabolism, anabolic hormone of the body. It regulates the metabol ...

. They are also

nerve cell A neuron (American English), neurone (British English), or nerve cell, is an excitable cell that fires electric signals called action potentials across a neural network in the nervous system. They are located in the nervous system and help to ...

survival factors. The BMPs bind to the bone morphogenetic protein receptor type-2 (BMPR2). They are involved in a multitude of cellular functions including osteogenesis,

, anterior/posterior axis specification, growth, and homeostasis. The TGFβ family includes:

TGFβ1 Transforming growth factor beta 1 or TGF-β1 is a polypeptide member of the transforming growth factor beta superfamily of cytokines. It is a secreted protein that performs many cellular functions, including the control of cell growth, cell proli ...

TGFβ2 Transforming growth factor-beta 2 (TGF-β2) is a secreted protein known as a cytokine that performs many cellular functions and has a vital role during embryonic development (alternative names: Glioblastoma-derived T-cell suppressor factor, G-TS ...

TGFβ3 Transforming growth factor beta-3 is a protein that in humans is encoded by the gene. It is a type of protein, known as a cytokine, which is involved in cell differentiation, embryogenesis and development. It belongs to a large family of cytokin ...

. Like the BMPs, TGFβs are involved not only in embryogenesis and cell differentiation, but also in apoptosis and other functions. They bind to TGF-beta receptor type-2 (TGFβR2).

binds to activin A receptor, type IIB

ACVR2B Activin receptor type-2B is a protein that in humans is encoded by the ''ACVR2B'' gene. ACVR2B is an activin type 2 receptor. Function Activins are dimeric growth and differentiation factors which belong to the transforming growth factor-bet ...

. It can then either form a receptor complex with activin A receptor, type IB (

ACVR1B Activin receptor type-1B is a protein that in humans is encoded by the ''ACVR1B'' gene. ACVR1B or ALK-4 acts as a transducer of activin or activin-like ligands (e.g., inhibin) signals. Activin binds to either ACVR2A or ACVR2B and then forms a c ...

) or with activin A receptor, type IC (

ACVR1C The activin A receptor also known as ACVR1C or ALK-7 is a protein that in humans is encoded by the ACVR1C gene. ACVR1C is a type I receptor for the TGFB family of signaling molecules. ACVR1C transduces signals of Nodal. Nodal binds to ACVR2B an ...

). When the receptor-ligand binding occurs via local action, this is classified as

paracrine signalling In cellular biology, paracrine signaling is a form of cell signaling, a type of cellular communication in which a cell produces a signal to induce changes in nearby cells, altering the behaviour of those cells. Signaling molecules known as para ...

Receptor recruitment and phosphorylation

The TGF beta ligand binds to a type II receptor dimer, which recruits a type I receptor dimer forming a hetero-tetrameric complex with the ligand. These receptors are serine/threonine kinase receptors. They have a

cysteine Cysteine (; symbol Cys or C) is a semiessential proteinogenic amino acid with the chemical formula, formula . The thiol side chain in cysteine enables the formation of Disulfide, disulfide bonds, and often participates in enzymatic reactions as ...

rich

extracellular domain An ectodomain is the domain of a membrane protein that extends into the extracellular space (the space outside a cell). Ectodomains are usually the parts of proteins that initiate contact with surfaces, which leads to signal transduction. A notabl ...

, a

transmembrane domain A transmembrane domain (TMD, TM domain) is a membrane-spanning protein domain. TMDs may consist of one or several alpha-helices or a transmembrane beta barrel. Because the interior of the lipid bilayer is hydrophobic, the amino acid residues in ...

, and a

cytoplasmic The cytoplasm describes all the material within a eukaryotic or prokaryotic cell, enclosed by the cell membrane, including the organelles and excluding the nucleus in eukaryotic cells. The material inside the nucleus of a eukaryotic cell and ...

serine/threonine rich domain. The

GS domain GS may stand for: Businesses and organizations * Goldman Sachs, one of the world's largest global investment banks * Global Star Software, a former Canadian video game publisher * GS (Swedish union), a trade union in Sweden * GS Group, a Korean ...

of the type I receptor consists of a series of about thirty

serine Serine (symbol Ser or S) is an α-amino acid that is used in the biosynthesis of proteins. It contains an α- amino group (which is in the protonated − form under biological conditions), a carboxyl group (which is in the deprotonated − ...

glycine Glycine (symbol Gly or G; ) is an amino acid that has a single hydrogen atom as its side chain. It is the simplest stable amino acid. Glycine is one of the proteinogenic amino acids. It is encoded by all the codons starting with GG (G ...

repeats. The binding of a TGFβ family ligand causes the rotation of the receptors so that their cytoplasmic kinase domains are arranged in a catalytically favorable orientation. The Type II receptor

residues of the Type I receptor, which activates the protein.

SMAD phosphorylation

There are five receptor regulated SMADs:

SMAD1 Mothers against decapentaplegic homolog 1 also known as SMAD family member 1 or SMAD1 is a protein that in humans is encoded by the ''SMAD1'' gene. Nomenclature SMAD1 belongs to the SMAD (protein), SMAD, a family of proteins similar to the gene p ...

SMAD2 Mothers against decapentaplegic homolog 2, also known as SMAD family member 2 or SMAD2, is a protein that in humans is encoded by the ''SMAD2'' gene. MAD homolog 2 belongs to the SMAD, a family of proteins similar to the gene products of the ''Dr ...

SMAD3 Mothers against decapentaplegic homolog 3 also known as SMAD family member 3 or SMAD3 is a protein that in humans is encoded by the SMAD3 gene. SMAD3 is a member of the SMAD (protein), SMAD family of proteins. It acts as a mediator of the signal ...

SMAD5 Mothers against decapentaplegic homolog 5 also known as SMAD5 is a protein that in humans is encoded by the ''SMAD5'' gene. SMAD5, as its name describes, is a homolog of the Drosophila gene: " Mothers against decapentaplegic", based on a traditi ...

, and

SMAD9 Mothers against decapentaplegic homolog 9 also known as SMAD9, SMAD8, and MADH6 is a protein that in humans is encoded by the ''SMAD9'' gene. SMAD9, as its name describes, is a homolog of the Drosophila gene: "Mothers against decapentaplegic". I ...

(sometimes referred to as SMAD8). There are essentially two intracellular pathways involving these

s. TGFβs, Activins, Nodals and some GDFs are mediated by SMAD2 and SMAD3, while BMPs, AMH and a few GDFs are mediated by

and

. The binding of the R-SMAD to the type I receptor is mediated by a zinc double finger

FYVE domain In molecular biology the FYVE zinc finger domain is named after the four cysteine-rich proteins: Fab 1 (yeast orthologue of PIKfyve), YOTB, Vac 1 (vesicle transport protein), and EEA1, in which it has been found. FYVE domains bind phosphatidyli ...

containing protein. Two such proteins that mediate the TGFβ pathway include SARA (the

SMAD anchor for receptor activation Zinc finger FYVE domain-containing protein 9 or SARA (SMAD anchor for receptor activation) is a protein that in humans is encoded by the ''ZFYVE9'' gene. SARA contains a double zinc finger ( FYVE domain). SARA is an anchoring protein involved in ...

) and HGS (Hepatocyte growth factor-regulated tyrosine kinase substrate). SARA is present in an early

endosome Endosomes are a collection of intracellular sorting organelles in eukaryotic cells. They are parts of the endocytic membrane transport pathway originating from the trans Golgi network. Molecules or ligands internalized from the plasma membra ...

which, by

clathrin-mediated endocytosis Receptor-mediated endocytosis (RME), also called clathrin-mediated endocytosis, is a process by which cells absorb metabolites, hormones, proteins – and in some cases viruses – by the inward budding of the plasma membrane (invagination). This ...

, internalizes the receptor complex.

SARA Sara may refer to: People * Sara (given name), a feminine given name People with the given name * Sara Aboobacker (1936–2023), Indian writer and translator * Sara Ahmed (born 1969), British-Australian writer * Sara Allgood (1880–1950), Ir ...

recruits an

. SARA permits the binding of the R-SMAD to the L45 region of the Type I receptor. SARA orients the R-SMAD such that serine residue on its

C-terminus The C-terminus (also known as the carboxyl-terminus, carboxy-terminus, C-terminal tail, carboxy tail, C-terminal end, or COOH-terminus) is the end of an amino acid chain (protein Proteins are large biomolecules and macromolecules that comp ...

faces the catalytic region of the Type I receptor. The Type I receptor

the serine residue of the R-SMAD. Phosphorylation induces a conformational change in the

MH2 domain MH or mH may refer to: Businesses and organizations * Malaysia Airlines, by IATA airline designator * Menntaskólinn við Hamrahlíð, a gymnasium in Reykjavík, Iceland * Miami Heat, an NBA basketball team Places * Mahalle, (abbreviated mh. on ...

of the R-SMAD and its subsequent dissociation from the receptor complex and SARA.

CoSMAD binding

The now phosphorylated RSMAD has high affinity for coSMAD (e.g.

) and forms a complex with one. The phosphate group does not act as a docking site for coSMAD, but rather the phosphorylation opens up an amino acid stretch allowing interaction.

Transcription

The phosphorylated RSMAD/coSMAD complex enters the nucleus where it binds transcription promoters/cofactors and causes the transcription of DNA. Bone morphogenetic proteins cause the transcription of

mRNA In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of Protein biosynthesis, synthesizing a protein. mRNA is ...

s involved in

osteogenesis Osteoblasts (from the Greek language, Greek combining forms for "bone", ὀστέο-, ''osteo-'' and βλαστάνω, ''blastanō'' "germinate") are cell (biology), cells with a single Cell nucleus, nucleus that synthesize bone. However, in the p ...

neurogenesis Neurogenesis is the process by which nervous system cells, the neurons, are produced by neural stem cells (NSCs). This occurs in all species of animals except the porifera (sponges) and placozoans. Types of NSCs include neuroepithelial cells ( ...

, and ventral mesoderm specification. TGFβs cause the transcription of mRNAs involved in

extracellular matrix In biology, the extracellular matrix (ECM), also called intercellular matrix (ICM), is a network consisting of extracellular macromolecules and minerals, such as collagen, enzymes, glycoproteins and hydroxyapatite that provide structural and bio ...

neogenesis and

immunosuppression Immunosuppression is a reduction of the activation or efficacy of the immune system. Some portions of the immune system itself have immunosuppressive effects on other parts of the immune system, and immunosuppression may occur as an adverse react ...

. They are also involved in G1 arrest in the

cell cycle The cell cycle, or cell-division cycle, is the sequential series of events that take place in a cell (biology), cell that causes it to divide into two daughter cells. These events include the growth of the cell, duplication of its DNA (DNA re ...

. Activin causes the transcription of mRNAs involved in

gonad A gonad, sex gland, or reproductive gland is a Heterocrine gland, mixed gland and sex organ that produces the gametes and sex hormones of an organism. Female reproductive cells are egg cells, and male reproductive cells are sperm. The male gon ...

al growth, embryo differentiation and placenta formation. Nodal causes the transcription of mRNAs involved in left and right axis specification,

mesoderm The mesoderm is the middle layer of the three germ layers that develops during gastrulation in the very early development of the embryo of most animals. The outer layer is the ectoderm, and the inner layer is the endoderm.Langman's Medical ...

and

endoderm Endoderm is the innermost of the three primary germ layers in the very early embryo. The other two layers are the ectoderm (outside layer) and mesoderm (middle layer). Cells migrating inward along the archenteron form the inner layer of the gastr ...

induction.

Pathway regulation

The TGF beta signaling pathway is involved in a wide range of cellular process and subsequently is very heavily regulated. There are a variety of mechanisms where the pathway is modulated either positively or negatively, including the agonists for ligands and R-SMADs, the decoy receptors, and the

ubiquitination Ubiquitin is a small (8.6 kDa) regulatory protein found in most tissues of eukaryotic organisms, i.e., it is found ''ubiquitously''. It was discovered in 1975 by Gideon Goldstein and further characterized throughout the late 1970s and 19 ...

of R-SMADs and receptors.

Ligand agonists/antagonists

Both

chordin Chordin (from Greek χορδή, string, catgut) is a protein with a prominent role in dorsal–ventral patterning during early embryonic development. In humans it is encoded for by the ''CHRD'' gene. History Chordin was originally identified ...

and noggin are

antagonists An antagonist is a character in a story who is presented as the main enemy or rival of the protagonist and is often depicted as a villain. It has been demonstrated that Chordin and Noggin dorsalize

. They are both found in the dorsal lip of ''

Xenopus ''Xenopus'' () (Gk., ξενος, ''xenos'' = strange, πους, ''pous'' = foot, commonly known as the clawed frog) is a genus of highly aquatic frogs native to sub-Saharan Africa. Twenty species are currently described with ...

'' and convert otherwise

epidermis The epidermis is the outermost of the three layers that comprise the skin, the inner layers being the dermis and Subcutaneous tissue, hypodermis. The epidermal layer provides a barrier to infection from environmental pathogens and regulates the ...

specified tissue into neural tissue (see

neurulation Neurulation refers to the folding process in vertebrate embryos, which includes the transformation of the neural plate into the neural tube. The embryo at this stage is termed the neurula. The process begins when the notochord induces the formati ...

). Noggin plays a key role in cartilage and bone patterning. Mice Noggin-/- have excess cartilage and lacked joint formation. Members of the DAN family of proteins also antagonize TGF beta family members. They include

Cerberus In Greek mythology, Cerberus ( or ; ''Kérberos'' ), often referred to as the hound of Hades, is a polycephaly, multi-headed dog that guards the gates of the Greek underworld, underworld to prevent the dead from leaving. He was the offspring o ...

, DAN, and

Gremlin A gremlin is a mischievous fictional creature invented at the beginning of the 20th century to originally explain malfunctions in aircraft, and later in other machinery, processes, and their operators. Depictions of these creatures vary widely. ...

. These proteins contain nine conserved

s which can form disulfide bridges. It is believed that DAN antagonizes

GDF5 Growth/differentiation factor 5 is a protein that in humans is encoded by the ''GDF5'' gene. The protein encoded by this gene is closely related to the bone morphogenetic protein (BMP) family and is a member of the TGF-beta superfamily. This gro ...

GDF6 Growth differentiation factor 6 (GDF6) is a protein that in humans is encoded by the ''GDF6'' gene. Function GDF6 belongs to the transforming growth factor beta superfamily and may regulate patterning of the ectoderm by interacting with bone m ...

and GDF7.

Follistatin Follistatin, also known as activin-bindings protein, is a protein that in humans is encoded by the ''FST'' gene. Follistatin is an autocrine glycoprotein that is expressed in nearly all tissues of higher animals. Its primary function is the b ...

inhibits Activin, which it binds. It directly affects

follicle-stimulating hormone Follicle-stimulating hormone (FSH) is a gonadotropin, a glycoprotein polypeptide hormone. FSH is synthesized and secreted by the gonadotropic cells of the anterior pituitary gland and regulates the development, growth, puberty, pubertal maturat ...

(FSH) secretion. Follistatin also is implicated in prostate cancers where mutations in its gene may preventing it from acting on activin which has anti-proliferative properties. Lefty is a regulator of TGFβ and is involved in the axis patterning during embryogenesis. It is also a member of the TGF superfamily of proteins. It is asymmetrically expressed in the left side of murine embryos and subsequently plays a role in left-right specification. Lefty acts by preventing the phosphorylation of R-SMADs. It does so through a constitutively active TGFβ type I receptor and through a process downstream of its activation. Drug-based antagonists have also been identified, such as SB431542, which selectively inhibits ALK4, ALK5, and ALK7.

Receptor regulation

The transforming growth factor receptor 3 (TGFβR3) is the most abundant of the TGF-β receptors yet, it has no known signaling domain. It however may serve to enhance the binding of TGFβ ligands to TGFβ type II receptors by binding TGFβ and presenting it to TGFβR2. One of the downstream targets of TGF β signaling, ''GIPC'', binds to its PDZ domain, which prevents its proteosomal degradation, which subsequently increases TGFβ activity. It may also serve as an

inhibin Activin and inhibin are two closely related protein complexes that have almost directly opposite biological effects. Identified in 1986, activin enhances FSH biosynthesis and secretion, and participates in the regulation of the menstrual c ...

coreceptor to ActivinRII. BMP and activin membrane bound inhibitor (BAMBI), has a similar extracellular domain as type I receptors. It lacks an intracellular serine/threonine protein kinase domain and hence is a pseudoreceptor. It binds to the type I receptor preventing it from being activated. It serves as a negative regulator of TGFβ signaling and may limit TGFβ expression during embryogeneis. It requires BMP signaling for its expression FKBP12 binds the GS region of the type I receptor preventing phosphorylation of the receptor by the type II receptors. It is believed that FKBP12 and its homologs help to prevent type I receptor activation in the absence of a ligands, since ligand binding causes its dissociation.

R-SMAD regulation

Role of inhibitory SMADs

There are two other SMADs which complete the SMAD family, the inhibitory SMADs (I-SMADS),

SMAD6 SMAD family member 6, also known as SMAD6, is a protein that in humans is encoded by the ''SMAD6'' gene. SMAD6 is a protein that, as its name describes, is a homolog of the Drosophila gene " mothers against decapentaplegic". It belongs to the SM ...

and SMAD7. They play a key role in the regulation of TGF beta signaling and are involved in negative feedback. Like other SMADs they have an MH1 and an MH2 domain. SMAD7 competes with other R-SMADs with the Type I receptor and prevents their phosphorylation. It resides in the nucleus and upon TGFβ receptor activation translocates to the cytoplasm where it binds the type I receptor.

binds SMAD4 preventing the binding of other R-SMADs with the coSMAD. The levels of I-SMAD increase with TGFβ signaling suggesting that they are downstream targets of TGFβ signaling.

R-SMAD ubiquitination

The E3 ubiquitin-protein

ligase In biochemistry, a ligase is an enzyme that can catalyze the joining ( ligation) of two molecules by forming a new chemical bond. This is typically via hydrolysis of a small pendant chemical group on one of the molecules, typically resulting i ...

SMURF1 E3 ubiquitin-protein ligase SMURF1 is an enzyme that in humans is encoded by the ''SMURF1'' gene. The SMURF1 Gene encodes a protein with a size of 757 amino acids and the molecular mass of this protein is 86114 Da. Function Smad ubiquitinatio ...

and

SMURF2 E3 ubiquitin-protein ligase SMURF2 is an enzyme that in humans is encoded by the ''SMURF2'' gene which is located at chromosome 17q23.3-q24.1. Interactions SMURF2 has been shown to interact with: * Mothers against decapentaplegic homolog 1, ...

regulate the levels of SMADs. They accept

ubiquitin Ubiquitin is a small (8.6 kDa) regulatory protein found in most tissues of eukaryotic organisms, i.e., it is found ''ubiquitously''. It was discovered in 1975 by Gideon Goldstein and further characterized throughout the late 1970s and 19 ...

from an E2 conjugating enzyme where they transfer ubiquitin to the RSMADs which causes their ubiquitination and subsequent proteosomal degradation. SMURF1 binds to

and

while SMURF2 binds

and SMAD7. It enhances the inhibitory action of SMAD7 while reducing the transcriptional activities of SMAD2.

Summary table

TGF-β ligands of ''H.sapiens'' highlighted in grey, of ''D.melanogaster'' ligands in pink, of ''C.elegans'' in yellow.

External links

* Kyoto Encyclopedia of Genes and Genomes
TGF beta signaling pathway
map * Netpath - A curated resource of signal transduction pathways in humans

References

{{TGFβ receptor superfamily modulators Cell signaling